Monitoring methods for novel insect-derived food: the PCR protocol for the detection and identification of Hermetia Illucens insects based on the HEI-COI probe and primer system

Abstract

Forwarding development of identification methods for novel foods, derived from edible insects, is necessary to ensure control over their marketing within the framework of the current legislation’s requirements.

The purpose of the research was the development and validation of a monoplex TaqMan-PCR assay protocol (a real-time polymerase chain reaction with TaqMan technology) for the insect Hermetia Illucens’ taxon-specific DNA detection and identification in food raw materials and foods.

Material and methods. Studies were performed using samples containing the target DNA sequence (dried whole larvae of H. Illucens as well as H. Illucens in oilcake meal and powdered capsule forms) and inherently not containing the target DNA sequence (other insect species, mammals, plants, microorganisms as well as multicomponent food: meat, dairy and plant food). DNA extraction and purification were performed by CTAB methods [commercial kits "Sorb-GMO-B" (Syntol, Russia) and "DNeasy mericon Food Kit" (QIAGEN, Germany)]. For amplification of the target sequence, which was a fragment of the cytochrome c oxidase subunit I mitochondrial gene, we used primers and the probe: Hei-COI-F (CCTGAGCTGGTATAGTGGGAAC); Hei-COI-R (AATTTGGTCATCTCCAATTAAGC); Hei-COI-P (FAM-CGAGCCGAATTAGGTCATCCAGG-BHQ-1). PCR conditions were optimized using CFX96TM Real-Time PCR System (Bio-Rad, USA) and Rotor-Gene Q (QIAGEN, Germany) amplifiers by empirical selection of primer and probe concentrations and amplification of the time/temperature profile. Specificity and limit of detection were evaluated as part of method validation.

Results and discussion. The optimized reaction mixture included 2.5-fold of Master Mix B [KCl, TrisCl (pH 8.8), 6.25 mM MgCl2], SynTaq DNA-polymerase, dNTP, glycerol, Tween 20, of each primers – 550 nM, probe – 100 nM. The time/temperature profile of the reaction: 95 °C – 180 s (95 °C – 15 s, 57 °C – 60 s), 40 cycles. The detection limit of the method was 0.19 ng of H. illucens DNA per reaction. The specificity of primer system and probe were experimentally confirmed in studies with DNA of other insects, animals, plants and microorganisms.

Conclusion. A protocol of a monoplex TaqMan-PCR assay for the taxon-specific DNA of insect Hermetia Illucens’ detection and identification in food raw materials and foods has been developed. Validity of the method has been confirmed by laboratory tests which allows to recommend it for use in surveillance of Hermetia Illucens-derived raw materials.

Keywords:DNA-based methods; control over novel food; edible insects; Hermetia Illucens; cytochrome c oxidase subunit I mitochondrial gene

Funding. The research was funded by the Russian Science Foundation (project № 20-16-00083).

Conflict of interest. The authors have no conflict of interest to declare.

Contribution. Concept and design of the study – Sadykova E.O., Tyshko N.V.; collecting and processing the material – Sadykova E.O., Shestakova S.I., Trebukh M.D., Nikitin N.S.; statistical processing – Sadykova E.O.; text writing – Sadykova E.O., Tyshko N.V.; editing, approval of the final version of the article, responsibility for the integrity of all parts of the article – all authors.

Acknowledgments. The authors sincerely thank the undergraduate of the Moscow Polytechnic University Ulyana O. Ryabinina and researcher of the Federal Research Centre of Nutrition, Biotechnology and Food Safety Nikita V. Trusov, for assistance in the researches’ carrying out.

For citation: Sadykova E.O., Tyshko N.V., Nikitin N.S., Trebukh M.D., Shestakova S.I. Monitoring methods for novel insect-derived food: the PCR protocol for the detection and identification of Hermetia Illucens insects based on the HEI-COI probe and primer system. Voprosy pitaniia [Problems of Nutrition]. 2023; 92 (1): 36–44. DOI: https://doi.org/10.33029/0042-8833-2023-92-1-36-44 (in Russian)

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CHIEF EDITOR
CHIEF EDITOR
Viktor A. Tutelyan
Full Member of the Russian Academy of Sciences, Doctor of Medical Sciences, Professor, Scientific Director of the Federal Research Centre of Nutrition, Biotechnology and Food Safety (Moscow, Russia)

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